Methods of protecting a plant from fungal pests

ABSTRACT

Phytoprotective compositions comprising one or more polypeptides having proteolytic activity in an agriculturally acceptable carrier, as well as methods of preparing such compositions and methods of using such compositions for preventing and/or treating phytopathogenic pests to reduce damage in plant propagation materials, plants and plant parts and increase crop yields.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/965,054, filed Jul. 27, 2020, which is a 35 U.S.C. 371 national stageentry of International Patent Application No. PCT/US2019/016860, filedFeb. 6, 2019, which claims priority to U.S. Provisional PatentApplication No. 62/626,940, filed Feb. 6, 2018. The disclosure of eachof the aforementioned applications is fully incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference. The name of the filecontaining the Sequence Listing is SQ.xml, which was created on Oct. 5,2023, and which has 6,772 bytes.

BACKGROUND

Because of increasing populations and corresponding demands for moreefficient and productive farms, there remains a need for new methods forprotecting crops and plants from disease and pests thereby preventingwaste and economic loss while improving crop yields and ensuring asufficient global food supply.

SUMMARY OF THE CLAIMED INVENTION

The present disclosure provides methods for of controlling or preventingpathogenic damage and/or pest damage in a plant propagation material, aplant, part of a plant and/or plant organ, comprising applying on theplant, part of the plant, plant organ, plant propagation material or asurrounding area thereof a phytoprotective agent comprising a protease.

DETAILED DESCRIPTION

This description is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented or of all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein, which do not depart from the instant invention, will be apparentto those skilled in the art in light of the instant disclosure. Hence,the following description is intended to illustrate some particularembodiments of the invention and not to exhaustively specify allpermutations, combinations and variations thereof.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andrelevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. For the sake of brevityand/or clarity, well-known functions or constructions may not bedescribed in detail.

As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

As used herein, the terms “acaricide” and “acaricidal” refer to an agentor combination of agents the application of which is toxic to an acarid(i.e., kills an acarid, inhibits the growth of an acarid and/or inhibitsthe reproduction of an acarid).

As used herein, the term “agriculturally beneficial agent” refers to anyagent (e.g., chemical or biological agent) or combination of agents theapplication of which causes or provides a beneficial and/or usefuleffect in agriculture including, but not limited to, agriculturallybeneficial microorganisms, biostimulants, nutrients, pesticides (e.g.,acaricides, fungicides, herbicides, insecticides, and nematicides) andplant signal molecules.

As used herein, the term “agriculturally beneficial microorganism”refers to a microorganism having at least one agriculturally beneficialproperty (e.g., the ability to fix nitrogen, the ability to solubilizephosphate and/or the ability to produce an agriculturally beneficialagent, such as a plant signal molecule).

As used herein, the term “agriculturally acceptable carrier” refers to asubstance or composition that can be used to deliver an agriculturallybeneficial agent to a plant, plant part or plant growth medium (e.g.,soil) without causing/having an unduly adverse effect on plant growthand/or yield. As used herein, the term “foliar-compatible carrier”refers to a material that can be foliarly applied to a plant or plantpart without causing/having an unduly adverse effect on the plant, plantpart, plant growth, plant health, or the like. As used herein, the term“seed-compatible carrier” refers to a material that can be applied to aseed without causing/having an unduly adverse effect on the seed, theplant that grows from the seed, seed germination, or the like. As usedherein, the term “soil-compatible carrier” refers to a material that canbe added to a soil without causing/having an unduly adverse effect onplant growth, soil structure, soil drainage, or the like.

As used herein, the term “and/or” is intended to include any and allcombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).Thus, the phrase “A, B and/or C” is to be interpreted as “A, A and B, Aand B and C, A and C, B, B and C, or C.”

As used herein, the terms “associated with,” “in association with” and“associated therewith,” when used in reference to a relationship betweena composition of the present disclosure and a plant or plant part, referto at least a juxtaposition or close proximity of the composition andthe plant or plant part. Such a juxtaposition or close proximity may beachieved by contacting or applying the composition directly to the plantor plant part and/or by applying the composition to the plant growthmedium (e.g., soil) in which the plant or plant part will be grown (oris currently being grown). According to some embodiments, thecomposition is applied as a coating to the outer surface of the plant orplant part. According to some embodiments, the composition is applied tosoil at, near or surrounding the site in which the plant or plant partwill be grown (or is currently being grown).

As used herein, the term “aqueous” refers to a composition that containsmore than a trace amount of water (i.e., more than 0.5% water by weight,based upon the total weight of the composition).

As used herein, the term “consists essentially of”, when used inreference to inoculant compositions and methods of the presentdisclosure, means that the compositions/methods may contain additionalcomponents/steps so long as the additional components/steps do notmaterially alter the composition/method. The term “materially alter,” asapplied to a composition/method of the present disclosure, refers to anincrease or decrease in the effectiveness of the composition/method ofat least 20%.

As used herein, the term “dispersant” refers to an agent or combinationof agents the application of which reduces the cohesiveness of likeparticles, the surface tension of a liquid, the interfacial tensionbetween two liquids and/or the interfacial tension between or a liquidand a solid.

As used herein, the terms “effective amount,” “effective concentration”and “effective amount/concentration” refer to an amount or concentrationthat is sufficient to cause a desired effect (e.g., reduced diseaseseverity). The absolute value of the amount/concentration that issufficient to cause the desired effect may be affected by factors suchas the type and magnitude of effect desired, the type, size and volumeof material to which the composition will be applied, the type(s) ofphytoprotective agent in the composition, the amount of phytoprotectiveagent in the composition, the stability of the phytoprotective agent inthe composition and the storage conditions (e.g., temperature, relativehumidity, duration). Those skilled in the art will understand how toselect an effective amount/concentration using routine dose-responseexperiments.

As used herein, the terms “enhanced growth” and “enhanced plant growth”refer to an improvement in one or more characteristics of plant growthand/or development as compared to one or more control plants (e.g., aplant germinated from an untreated seed or an untreated plant).Exemplary plant growth/development characteristics include, but are notlimited to, biomass, carbohydrate biosynthesis, chlorophyll content,cold tolerance, drought tolerance, height, leaf length, leaf mass, leafnumber, leaf surface area, leaf volume, nutrient uptake (e.g., calcium,iron, magnesium, nitrogen, phosphorous, potassium and/or sulfur uptake),rate(s) of photosynthesis, root area, root diameter, root length, rootmass, root nodulation (e.g., nodule mass, nodule number, nodule volume),root number, root surface area, root volume, salt tolerance, seedgermination, seedling emergence, shoot diameter, shoot length, shootmass, shoot number, shoot surface area, shoot volume, spread, stomatalconductance and survival rate. Unless otherwise indicated, references toenhanced plant growth are to be interpreted as meaning that compositionsand methods of the present disclosure enhance plant corn growth bycontrolling or preventing pathogenic damage and/or pest damage in aplant propagation material, a plant, part of a plant and/or plant organ,enhancing nutrient availability, improving soil characteristics, etc.and are not to be interpreted as suggesting that compositions andmethods of the present disclosure act as plant growth regulators.

As used herein, the terms “enhanced yield” and “enhanced plant yield”refer to an improvement in one or more characteristics of plant yield ascompared to one or more control plants (e.g., a control plant germinatedfrom an untreated seed). Exemplary plant yield characteristics include,but are not limited to, biomass; bushels per acre; grain weight per plot(GWTPP); nutritional content; percentage of plants in a given area(e.g., plot) that fail to produce grain; yield at standard moisturepercentage (YSMP), such as grain yield at standard moisture percentage(GYSMP); yield per plot (YPP), such as grain weight per plot (GWTPP);and yield reduction (YRED). Unless otherwise indicated, references toenhanced plant yield are to be interpreted as meaning that compositionsand methods of the present disclosure enhance plant yield by controllingor preventing pathogenic damage and/or pest damage in a plantpropagation material, a plant, part of a plant and/or plant organ,enhancing nutrient availability, improving soil characteristics, etc.and are not to be interpreted as suggesting that compositions andmethods of the present disclosure act as plant growth regulators.

As used herein, the term “foliage” refers to those portions of a plantthat normally grow above the ground, including, but not limited to,leaves, stalks, stems, flowers, fruiting bodies and fruits.

As used herein, the terms “foliar application” and “foliarly applied”refer to the application of the composition of the present disclosure tothe foliage of a plant (e.g., to the leaves of the plant). Applicationmay be effected by any suitable means, including, but not limited to,spraying the plant with the composition of the present disclosure. Insome embodiments, the composition of the present disclosure is/areapplied to the leaves, stems and/or stalk of the plant and not to theflowers, fruiting bodies or fruits of the plant.

As used herein, the terms “fungicide” and “fungicidal” refer to an agentor combination of agents the application of which is toxic to a fungus(i.e., kills a fungus, inhibits the growth of a fungus and/or inhibitsthe reproduction of a fungus).

As used herein, the terms “herbicide” and “herbicidal” refer to an agentor combination of agents the application of which is toxic to a weed(i.e., kills a weed, inhibits the growth of a weed and/or inhibits thereproduction of a weed).

As used herein, the terms “insecticide” and “insecticidal” refer to anagent or combination of agents the application of which is toxic to aninsect (i.e., kills an insect, inhibits the growth of an insect and/orinhibits the reproduction of an insect).

As used herein, the term “isomer” includes all stereoisomers of thecompounds and/or molecules to which it refers, including enantiomers anddiastereomers, as well as all conformers, roatmers and tautomers, unlessotherwise indicated. Compounds and/or molecules disclosed herein includeall enantiomers in either substantially pure levorotatory ordextrorotatory form, or in a racemic mixture, or in any ratio ofenantiomers. Where embodiments disclose a (D)-enantiomer, thatembodiment also includes the (L)-enantiomer; where embodiments disclosea (L)-enantiomer, that embodiment also includes the (D)-enantiomer.Where embodiments disclose a (+)-enantiomer, that embodiment alsoincludes the (−)-enantiomer; where embodiments disclose a(−)-enantiomer, that embodiment also includes the (+)-enantiomer. Whereembodiments disclose a (S)-enantiomer, that embodiment also includes the(R)-enantiomer; where embodiments disclose a (R)-enantiomer, thatembodiment also includes the (S)-enantiomer. Embodiments are intended toinclude any diastereomers of the compounds and/or molecules referred toherein in diastereomerically pure form and in the form of mixtures inall ratios. Unless stereochemistry is explicitly indicated in a chemicalstructure or chemical name, the chemical structure or chemical name isintended to embrace all possible stereoisomers, conformers, rotamers andtautomers of compounds and/or molecules depicted.

As used herein, the terms “nematicide” and “nematicidal” refer to anagent or combination of agents the application of which is toxic to anematode (i.e., kills a nematode, inhibits the growth of a nematodeand/or inhibits the reproduction of a nematode).

As used herein, the term “nitrogen fixing organism” refers to anorganism capable of converting atmospheric nitrogen (N₂) into a formthat may be utilized by a plant or plant part (e.g., ammonia (NH₃),ammonium (NH₄ ⁺), etc.).

As used herein, the term “non-aqueous” refers to a composition thatcomprises no more than a trace amount of water (i.e., no more than 0.5%water by weight, based upon the total weight of the composition).

As used herein, the term “nutrient” refers to a compound or elementuseful for nourishing a plant (e.g., vitamins, macrominerals,micronutrients, trace minerals, organic acids, etc. that are necessaryfor plant growth and/or development).

As used herein, the terms “percent identity,” “% identity” and “percentidentical” refer to the relatedness of two or more nucleotide or aminoacid sequences, which may be calculated by (i) comparing two optimallyaligned sequences over a window of comparison, (ii) determining thenumber of positions at which the identical nucleic acid base (fornucleotide sequences) or amino acid residue (for proteins) occurs inboth sequences to yield the number of matched positions, (iii) dividingthe number of matched positions by the total number of positions in thewindow of comparison, and then (iv) multiplying this quotient by 100% toyield the percent identity. If the “percent identity” is beingcalculated in relation to a reference sequence without a particularcomparison window being specified, then the percent identity isdetermined by dividing the number of matched positions over the regionof alignment by the total length of the reference sequence. Accordingly,for purposes of the present invention, when two sequences (query andsubject) are optimally aligned (with allowance for gaps in theiralignment), the “percent identity” for the query sequence is equal tothe number of identical positions between the two sequences divided bythe total number of positions in the query sequence over its length (ora comparison window), which is then multiplied by 100%.

As used herein, the term “pest” includes any organism or virus thatnegatively affects a plant, including, but not limited to, organisms andviruses that spread disease, damage host plants and/or compete for soilnutrients. The term “pest” encompasses organisms and viruses that areknown to associate with plants and to cause a detrimental effect on theplant's health and/or vigor. Plant pests include, but are not limitedto, microbial pests, preferably bacteria and/or fungi, includingoomycetes.

As used herein, the terms “pesticide” and “pesticidal” refer to agentsor combinations of agents the application of which is toxic to a pest(i.e., kills a pest, inhibits the growth of a pest and/or inhibits thereproduction of a pest). Non-limiting examples of pesticides includeacaricides, fungicides, herbicides, insecticides, and nematicides, etc.

As used herein, the term “plant” includes all plant populations,including, but not limited to, agricultural, horticultural andsilvicultural plants. The term “plant” encompasses plants obtained byconventional plant breeding and optimization methods (e.g.,marker-assisted selection) and plants obtained by genetic engineering,including cultivars protectable and not protectable by plant breeders'rights.

As used herein, the term “plant cell” refers to a cell of an intactplant, a cell taken from a plant, or a cell derived from a cell takenfrom a plant. Thus, the term “plant cell” includes cells within seeds,suspension cultures, embryos, meristematic regions, callus tissue,leaves, shoots, gametophytes, sporophytes, pollen and microspores.

As used herein, the term “plant part” refers to any part of a plant,including cells and tissues derived from plants. Thus, the term “plantpart” may refer to any of plant components or organs (e.g., leaves,stems, roots, etc.), plant tissues, plant cells and seeds. Examples ofplant parts, include, but are not limited to, anthers, embryos, flowers,fruits, fruiting bodies, leaves, ovules, pollen, rhizomes, roots, seeds,shoots, stems and tubers, as well as scions, rootstocks, protoplasts,calli and the like.

As used herein, the term “plant propagation material” refers to a plantpart from which a whole plant can be generated. Examples of plantpropagation materials include, but are not limited to, cuttings (e.g.,leaves, stems), rhizomes, seeds, tubers and cells/tissues that can becultured into a whole plant.

As used herein, the term “reduced disease severity” refers to ameasurable reduction in the relative or absolute percentage orproportion of a sample showing symptoms of disease.

While certain aspects of the present disclosure will hereinafter bedescribed with reference to embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present disclosure as defined by the claims.

Compositions of the present disclosure may comprise any agriculturallyacceptable carrier(s), including, but not limited to, foliar-compatiblecarriers, seed-compatible carriers and soil-compatible carriers.Selection of appropriate carrier materials will depend on the intendedapplication(s) and the elements present in the composition. In someembodiments, the carrier material(s) will be selected to provide acomposition in the form of a liquid, gel, slurry, or solid. In someembodiments, the carrier will consist essentially of or consist of oneor more stabilizing compounds.

In some embodiments, the composition comprises one or more solidcarriers. According to some embodiments, the composition comprises oneor more powders (e.g., wettable powders) and/or granules. Non-limitingexamples of solid carriers include clays (e.g., attapulgite clays,montmorillonite clay, etc.), peat-based powders and granules,freeze-dried powders, spray-dried powders, spray-freeze-dried powdersand combinations thereof. In some embodiments, the composition comprisesone or more liquid and/or gel carriers. According to some embodiments,the composition comprises one or more non-aqueous solvents. According tosome embodiments, the composition comprises one or more aqueous solvents(e.g., water).

Compositions of the present disclosure comprising non-dusting granulatesmay be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and4,661,452 and may optionally be coated by methods known in the art.Examples of waxy coating materials are poly(ethylene oxide) products(polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000;ethoxylated nonylphenols having from 16 to 50 ethylene oxide units;ethoxylated fatty alcohols in which the alcohol contains from 12 to 20carbon atoms and in which there are 15 to 80 ethylene oxide units; fattyalcohols; fatty acids; and mono- and di- and triglycerides of fattyacids. Examples of film-forming coating materials suitable forapplication by fluid bed techniques are given in GB 1483591.Compositions of the present disclosure comprising liquid enzymepreparations may, for instance, be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acidaccording to established methods. Compositions of the present disclosurecomprising enzymes may be prepared according to the method disclosed inEP 238,216.

Compositions of the present disclosure may be formulated as a granulefor example as a co-granule that combines one or more enzymes. Eachenzyme will then be present in more granules securing a more uniformdistribution of enzymes in, for example, a detergent. This also reducesthe physical segregation of different enzymes due to different particlesizes.

An embodiment of the composition of the present disclosure relates to anenzyme granule/particle comprising an enzyme. The granule is composed ofa core, and optionally one or more coatings (outer layers) surroundingthe core. Typically, the granule/particle size, measured as equivalentspherical diameter (volume based average particle size), of the granuleis 20-2000 μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm.

The core may include additional materials such as fillers, fibrematerials (cellulose or synthetic fibres), stabilizing agents,solubilizing agents, suspension agents, viscosity regulating agents,light spheres, plasticizers, salts, lubricants and fragrances. The coremay include binders, such as synthetic polymer, wax, fat, orcarbohydrate. The core may comprise a salt of a multivalent cation, areducing agent, an antioxidant, a peroxide decomposing catalyst and/oran acidic buffer component, typically as a homogenous blend. The coremay consist of an inert particle with an enzyme absorbed into it, orapplied onto the surface, e.g., by fluid bed coating. The core may havea diameter of 20-2000 μm, particularly 50-1500 μm, 100-1500 μm or250-1200 μm. The core can be prepared by granulating a blend of theingredients, e.g., by a method comprising granulation techniques such ascrystallization, precipitation, pan-coating, fluid bed coating, fluidbed agglomeration, rotary atomization, extrusion, prilling,spheronization, size reduction methods, drum granulation, and/or highshear granulation.

Methods for preparing the core can be found in Handbook of PowderTechnology; Particle size enlargement by C. E. Capes; Volume 1; 1980;Elsevier. Preparation methods include known feed and granule formulationtechnologies, e.g.:

a) Spray dried products, wherein a liquid enzyme-containing solution isatomized in a spray drying tower to form small droplets which duringtheir way down the drying tower dry to form an enzyme-containingparticulate material. Very small particles can be produced this way(Michael S. Showell (editor); Powdered detergents; Surfactant ScienceSeries; 1998; vol. 71; page 140-142; Marcel Dekker).

b) Layered products, wherein the enzyme is coated as a layer around apre-formed inert core particle, wherein an enzyme-containing solution isatomized, typically in a fluid bed apparatus wherein the pre-formed coreparticles are fluidized, and the enzyme-containing solution adheres tothe core particles and dries up to leave a layer of dry enzyme on thesurface of the core particle. Particles of a desired size can beobtained this way if a useful core particle of the desired size can befound. This type of product is described in, e.g., WO 97/23606

c) Absorbed core particles, wherein rather than coating the enzyme as alayer around the core, the enzyme is absorbed onto and/or into thesurface of the core. Such a process is described in WO 97/39116.

d) Extrusion or pelletized products, wherein an enzyme-containing pasteis pressed to pellets or under pressure is extruded through a smallopening and cut into particles which are subsequently dried. Suchparticles usually have a considerable size because of the material inwhich the extrusion opening is made (usually a plate with bore holes)sets a limit on the allowable pressure drop over the extrusion opening.Also, very high extrusion pressures when using a small opening increaseheat generation in the enzyme paste, which is harmful to the enzyme (seealso Michael S. Showell (editor); Powdered detergents; SurfactantScience Series; 1998; vol. 71; page 140-142; Marcel Dekker).

e) Prilled products, wherein an enzyme-containing powder is suspended inmolten wax and the suspension is sprayed, e.g., through a rotating diskatomiser, into a cooling chamber where the droplets quickly solidify(Michael S. Showell (editor); Powdered detergents; Surfactant ScienceSeries; 1998; vol. 71; page 140-142; Marcel Dekker). The productobtained is one wherein the enzyme is uniformly distributed throughoutan inert material instead of being concentrated on its surface. Inaddition, U.S. Pat. Nos. 4,016,040 and 4,713,245 are documents relatingto this technique.

f) Mixer granulation products, wherein a liquid is added to a dry powdercomposition of, e.g., conventional granulating components, the enzymebeing introduced either via the liquid or the powder or both. The liquidand the powder are mixed and as the moisture of the liquid is absorbedin the dry powder, the components of the dry powder will start to adhereand agglomerate and particles will build up, forming granulatescomprising the enzyme. Such a process is described in U.S. Pat. No.4,106,991 and related documents EP 170360, EP 304332, EP 304331, WO90/09440 and WO 90/09428. In a particular product of this processwherein various high- shear mixers can be used as granulators,granulates consisting of enzyme as enzyme, fillers and binders etc. aremixed with cellulose fibres to reinforce the particles to give theso-called T-granulate. Reinforced particles, being more robust, releaseless enzymatic dust.

g) Size reduction, wherein the cores are produced by milling or crushingof larger particles, pellets, tablets, briquettes etc. containing theenzyme. The wanted core particle fraction is obtained by sieving themilled or crushed product. Over and undersized particles can berecycled. Size reduction is described in (Martin Rhodes (editor);Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons).

h) Fluid bed granulation. Fluid bed granulation involves suspendingparticulates in an air stream and spraying a liquid onto the fluidizedparticles via nozzles. Particles hit by spray droplets get wetted andbecome tacky. The tacky particles collide with other particles andadhere to them and form a granule.

i) The cores may be subjected to drying, such as in a fluid bed drier.The drying preferably takes place at a product temperature of from 25 to90° C. For some embodiments of the composition of the present disclosurecomprising an enzyme it is important the cores comprising the enzymecontain a low amount of water before coating. If water sensitive enzymesare coated before excessive water is removed, it will be trapped withinthe core and it may affect the activity of the enzyme negatively. Afterdrying, the cores preferably contain 0.1-10% w/w water.

The core of the enzyme granule/particle may be surrounded by at leastone coating, e.g., to improve the storage stability, to reduce dustformation during handling, or for coloring the granule. The optionalcoating(s) may include a salt coating, or other suitable coatingmaterials, such as polyethylene glycol (PEG), methyl hydroxy-propylcellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzymegranules with multiple coatings are shown in WO 93/07263 and WO97/23606.

The coating may be applied in an amount of at least 0.1% by weight ofthe core, e.g., at least 0.5%, 1% or 5%. The amount may be at most 100%,70%, 50%, 40% or 30%. The coating is preferably at least 0.1 μm thick,particularly at least 0.5 μm, at least 1 μm or at least 5 μm. In aparticular embodiment the thickness of the coating is below 100 μm. In amore particular embodiment the thickness of the coating is below 60 μm.In an even more particular embodiment the total thickness of the coatingis below 40 μm. The coating should encapsulate the core unit by forminga substantially continuous layer. A substantially continuous layer is tobe understood as a coating having few or no holes, so that the core unitit is encapsulating/enclosing has few or none uncoated areas. The layeror coating should in particular be homogeneous in thickness. The coatingcan further contain other materials as known in the art, e.g., fillers,antisticking agents, pigments, dyes, plasticizers and/or binders, suchas titanium dioxide, kaolin, calcium carbonate or talc.

A salt coating may comprise at least 60% by weight w/w of a salt, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% by weight w/w. The salt may beadded from a salt solution where the salt is completely dissolved orfrom a salt suspension wherein the fine particles is less than 50 μm,such as less than 10 μm or less than 5 μm. The salt coating may comprisea single salt or a mixture of two or more salts. The salt may be watersoluble, in particular having a solubility at least 0.1 grams in 100 gof water at 20° C., preferably at least 0.5 g per 100 g water, e.g., atleast 1 g per 100 g water, e.g., at least 5 g per 100 g water.

The salt may be an inorganic salt, e.g., salts of sulfate, sulfite,phosphate, phosphonate, nitrate, chloride or carbonate or salts ofsimple organic acids (less than 10 carbon atoms, e.g., 6 or less carbonatoms) such as citrate, malonate or acetate. Examples of cations inthese salts are alkali or earth alkali metal ions, the ammonium ion ormetal ions of the first transition series, such as sodium, potassium,magnesium, calcium, zinc or aluminium. Examples of anions includechloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate,phosphate, monobasic phosphate, dibasic phosphate, hypophosphite,dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate,metasilicate, citrate, malate, maleate, malonate, succinate, lactate,formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate orgluconate. In particular, alkali- or earth alkali metal salts ofsulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonateor salts of simple organic acids such as citrate, malonate or acetatemay be used.

The salt in the coating may have a constant humidity at 20° C. above60%, particularly above 70%, above 80% or above 85%, or it may beanother hydrate form of such a salt (e.g., anhydrate). The salt coatingmay be as described in WO 00/01793 or WO 2006/034710.

Specific examples of suitable salts are NaCl (CH_(20° C.)=76%), Na₂CO₃(CH_(20° C.)=92%), NaNO₃ (CH_(20° C.)=73%), Na₂HPO₄ (CH_(20° C.)=95%),Na₃PO₄ (CH_(25° C.)=92%), NH₄Cl (CH_(20° C)=79.5%), (NH₄)₂HPO₄(CH_(20° C.)=93.0%), NH₄H₂PO₄ (CH_(20° C.) =93.1%), (NH₄)₂SO₄(CH_(20° C.)=81.1%), KCl (CH_(20° C.)=85%), K₂HPO₄ (CH_(20° C)=92%),KH₂PO₄(CH_(20° C.)=96.5%), KNO₃ (CH_(20° C)=93.5%),Na₂SO₄(CH_(20° C.)=93%), K₂SO₄ (CH_(20° C)=98%), KHSO₄(CH_(20° C.)=86%), MgSO₄ (CH_(20° C.)=90%), ZnSO₄ (CH_(20° C.)=90%) andsodium citrate (CH_(25° C.)=86%). Other examples include NaH₂PO₄,(NH₄)H₂PO₄, CuSO₄, Mg(NO₃)₂ and magnesium acetate.

The salt may be in anhydrous form, or it may be a hydrated salt, i.e. acrystalline salt hydrate with bound water(s) of crystallization, such asdescribed in WO 99/32595. Specific examples include anhydrous sodiumsulfate (Na₂SO₄), anhydrous magnesium sulfate (MgSO₄), magnesium sulfateheptahydrate (MgSO₄·7H₂O), zinc sulfate heptahydrate (ZnSO₄·7H₂O),sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O), magnesium nitratehexahydrate (Mg(NO₃)₂(6H₂O)), sodium citrate dihydrate and magnesiumacetate tetrahydrate. Preferably the salt is applied as a solution ofthe salt, e.g., using a fluid bed.

Thus, in a further aspect, a composition used in the methods of thepresent disclosure comprises a granule, which comprises: (a) a corecomprising an enzyme, and (b) optionally a coating consisting of one ormore layer(s) surrounding the core.

In some embodiments, compositions of the present disclosure are appliedat a rate of about to about 100 milliliters and/or grams of compositionper plant. According to some embodiments, one or more compositions ofthe present disclosure is/are applied in an amount sufficient to ensureeach plant is treated with about/at least 0.05, 0.1, 0.125, 0.15, 0.175,0.2, 0.2.5, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475,0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2,2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100milliliters and/or grams of inoculant composition. According to someembodiments, one or more compositions of the present disclosure is/areapplied in an amount sufficient to ensure that an average of about/atleast 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.2.5, 0.275, 0.3, 0.325,0.35, 0.375, 0.4, 0.425, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8,0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5,3.75, 4, 4.25, 4.5, 4.75 or 5 milliliters and/or grams of composition isapplied to each plant.

In some embodiments, compositions of the present disclosure are appliedat a rate of about 0.5 to about 100 milliliters and/or grams ofcomposition per acre of treated crops. According to some embodiments,one or more compositions of the present disclosure is/are applied in anamount sufficient to ensure each acre of treated crops is treated withabout/at least 0.05, 0.1, 0.15, 0.175, 0.2, 0.225, 0.2.5, 0.275, 0.3,0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.6, 0.65, 0.7, 0.75,0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25,3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 milliliters and/or grams ofcomposition. According to some embodiments, one or more compositions ofthe present disclosure is/are applied in an amount sufficient to ensurethat an average of about/at least 0.05, 0.1, 0.125, 0.15, 0.175, 0.2,0.225, 0.2.5, 0.275, 0.3, 0.325, 0.35, 0.4, 0.425, 0.45, 0.475, 0.5,0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2,2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75 or 5 millilitersand/or grams of composition is applied to each acre of treated crops.

In some embodiments, compositions of the present disclosure are appliedat a rate of about to about 100 milliliters and/or grams of compositionper acre of plant growth media. According to some embodiments, one ormore compositions of the present disclosure is/are applied in an amountsufficient to ensure each acre of plant growth media is treated withabout/at least 0.05, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.2.5, 0.275,0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.475, 0.5, 0.55, 0.6, 0.65, 0.7,0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3,3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 20, 30, 40, 50, 70, 80, 90 or 100 milliliters and/or grams ofcomposition. According to some embodiments, one or more compositions ofthe present disclosure is/are applied in an amount sufficient to ensurethat an average of about/at least 0.05, 0.1, 0.125, 0.15, 0.175, 0.2,0.225, 0.2.5, 0.275, 0.3, 0.325, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5,0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2,2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 20 , 30, 40, 50, 100, 500, 1,000, 5,000, or 10,000 milliliters and/or grams ofcomposition is applied to each acre of plant growth media.

In some embodiments, the composition comprises a protease derived from astrain selected from the group Alicyclobacillus, Arthrobacter,Aspergillus (such as Aspergillus oryzae), Bacillus (such as, Bacillusamyloliquefaciens, Bacillus clausii, Bacillus licheniformis, Bacillusmojavensis, and Bacillus pumilus), Dichomitus squalens, Fusariumoxysporum, Janibacter, Lysobacter, Meripilus giganteus, Nocardiopsisprasina, Pyrococcus furiosus, Rhizomucor miehei, Saccharomonosporaviridis, Saccharothrix australiensis, Saccharothrix variisporea,Streptomyces (such as Streptomyces violaceoruber), Streptosporangiumalbidum, Thermoascus aurantiacus, Trichoderma reesei, and Zophobasatratus.

The present invention is further described by the following numberedparagraphs:

Paragraph [1]. A method comprising foliar application of a compositioncomprising an effective amount of a phytoprotective agent to a plantand/or plant part.

Paragraph [2]. A method of controlling or preventing one or more plantdiseases and/or plant pests in a plant or plant part and/or inducingdisease resistance to a pathogen in a plant or plant part, comprisingapplying an effective amount of a composition comprising aphytoprotective agent to the plant or plant part.

Paragraph [3]. The method of controlling or preventing pathogenic damageand/or pest damage in a plant propagation material, a plant, part of aplant and/or plant organ, comprising applying on the plant, part of theplant, plant organ, plant propagation material or a surrounding areathereof an effective amount of a composition comprising aphytoprotective agent.

Paragraph [4]. The method of any one of paragraphs 1-3, wherein thephytoprotection agent comprises a protease.

Paragraph [5]. The method of any one of paragraphs 1-3, wherein thephytoprotective agent comprises a polypeptide having at least 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 97.5%, 98% 98.5%, 99%, 99.5%, or 100% sequenceidentity with a sequence selected from the group SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

Paragraph [6]. The method of any one of paragraphs 1-3, wherein thephytoprotective agent comprises a protease derived from a strainselected from the group Alicyclobacillus, Arthrobacter, Aspergillusoryzae, Bacillus (such as, Bacillus amyloliquefaciens, Bacillus clausii,Bacillus licheniformis, Bacillus mojavensis, and Bacillus pumilus),Dichomitus squalens, Fusarium oxysporum, Janibacter, Lysobacter,Meripilus giganteus, Nocardiopsis prasina, Pyrococcus furiosus,Rhizomucor miehei, Saccharomonospora viridis, Saccharothrixaustraliensis, Saccharothrix variisporea, Streptomyces (such asStreptomyces violaceoruber), Streptosporangium albidum, Thermoascusaurantiacus, Trichoderma reesei, and Zophobas atratus.

Paragraph [7]. The method of any one of paragraphs 1-3, wherein thephytoprotective agent comprises an endopeptidase.

Paragraph [8]. The method of any one of paragraphs 1-7, wherein theplant part is foliage.

Paragraph [9]. The method of any one of paragraphs 1-8, wherein thephytoprotective agent is in an amount or concentration of about 0.0001to about 95% or more (by weight) of the composition, for example about0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.001,0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055,0.006, 0.0065, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02,0.025, 0.03, 0.035, 0.04, 0.045, 0.06, 0.07, 0.08, 0.09, 0.1, 0.02, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 to about 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4.,4.5, 4.6, 4.7, 4.8, 4.9, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 65,70, 75, 80, 85, 90, 95% (by weight) of the composition.

Paragraph [10]. The method of any one of paragraphs 1-9, wherein thecomposition further comprises an agriculturally acceptable carrier, forexample, a foliar-compatible carrier, a seed-compatible carrier, and/ora soil-compatible carrier, wherein the carrier comprises a liquid, gel,slurry, or solid, optionally:

one or more monosaccharides, optionally arabinose, fructose and/orglucose;

one or more disaccharides, optionally maltose, sucrose and/or trehalose;

one or more maltodextrins, optionally one or more maltodextrins (e.g.,one or more maltodextrins (each and/or collectively) having a DEV valueof about 15 to about 20;

one or more sugar alcohols, optionally arabitol, mannitol, sorbitoland/or xylitol;

one or more humic acids, optionally potassium humate and/or sodiumhumate;

one or more fulvic acids, optionally potassium fulvate and/or sodiumfulvate;

one or more hygroscopic polymers, optionally one or more albumins,alginates, celluloses, gums (e.g., cellulose gum, guar gum, gum arabic,gum combretum, xantham gum), methyl celluloses, nylons, pectins,polyacrylic acids, polycarbonates, polyethylene glycols (PEG),polyethylenimines (PEI), polylactides, polymethylacrylates (PMA),polyurethanes, polyvinyl alcohols (PVA), polyvinylpyrrolidones (PVP),propylene glycols, sodium carboxymethyl celluloses and/or starches;

one or more oxidation control components, optionally one or moreantioxidants (e.g., ascorbic acid, ascorbyl palmitate, ascorbylstearate, calcium ascorbate, one or more carotenoids, lipoic acid, oneor more phenolic compounds (e.g., one or more flavonoids, flavonesand/or flavonols), potassium ascorbate, sodium ascorbate, one or morethiols (e.g., glutathione, lipoic acid and/or N-acetyl cysteine), one ormore tocopherols, one or more tocotrienols, ubiquinone and/or uric acid)and/or one or more oxygen scavengers, optionally ascorbic acid and/orsodium hydrogen carbonate; and/or

one or more UV protectants, optionally one or more lignosulfites.

Paragraph [11]. The method of any one of paragraphs 1-10, wherein thecomposition further comprises one or more pesticides, optionally:

one or more acaricides, insecticides and/or nematicides, optionally oneor more carbamates, diamides, macrocyclic lactones, neonicotinoids,organophosphates, phenylpyrazoles, pyrethrins, spinosyns, syntheticpyrethroids, tetronic acids and/or tetramic acids;

one or more fungicides, optionally one or more aromatic hydrocarbons,benzimidazoles, benzthiadiazole, carboxamides, carboxylic acid amides,morpholines, phenylamides, phosphonates, quinone outside inhibitors,thiazolidines, thiophanates, thiophene carboxamides and/or triazoles;

one or more gastropodicides, optionally one or more iron phosphates,metaldehydes, methiocarbs and/or salts;

one or more herbicides, optionally one or more acetyl CoA carboxylase(ACCase) inhibitors, acetolactate synthase (ALS) inhibitors,acetohydroxy acid synthase (AHAS) inhibitors, photosystem II inhibitors,photosystem I inhibitors, protoporphyrinogen oxidase (PPO or Protox)inhibitors, carotenoid biosynthesis inhibitors, enolpyruvylshikimate-3-phosphate (EPSP) synthase inhibitor, glutamine synthetaseinhibitor, dihydropteroate synthetase inhibitor, mitosis inhibitors,4-hydroxyphenyl-pyruvate-dioxygenase (4-HPPD) inhibitors, syntheticauxins, auxin herbicide salts, auxin transport inhibitors, and/ornucleic acid inhibitors;

one or more rodenticides, optionally brodifacoum, bromadiolone,bromethalin, cholecalciferol, chlorophacinone, difethialone,diphacinone, strychnine, warfarin and/or zinc phosphide; and/or

one or more virucides.

Paragraph [12]. The method of any one of paragraphs 1-11, wherein thecomposition further comprises one or more flavonoids, optionally:

one or more anthocyanidins, optionally cyanidin, delphinidin, malvidin,pelargonidin, peonidin and/or petunidin;

one or more anthoxanthins, optionally one or more flavones, such asapigenin, baicalein, chrysin, 7,8-dihydroxyflavone, diosmin, flavoxate,6-hydroxyflavone, luteolin, scutellarein, tangeritin and/or wogonin;and/or flavonols, such as amurensin, astragalin, azaleatin, azalein,fisetin, furanoflavonols galangin, gossypetin, 3-hydroxyflavone,hyperoside,icariin, isoquercetin, kaempferide, kaempferitrin,kaempferol, isorhamnetin, morin, myricetin, myricitrin, natsudaidain,pachypodol, pyranoflavonols quercetin, quericitin, rhamnazin, rhamnetin,robinin, rutin, spiraeoside, troxerutin and/or zanthorhamnin;

one or more flavanones, optionally butin, eriodictyol, hesperetin,hesperidin, homoeriodictyol, isosakuranetin, naringenin, naringin,pinocembrin, poncirin, sakuranetin, sakuranin and/or sterubin;

one or more flavanonols, optionally dihydrokaempferol and/or taxifolin;flavans, such as flavan-3-ols (e.g., catechin (C), catechin 3-gallate(Cg), epicatechins (EC), epigallocatechin (EGC) epicatechin 3-gallate(ECg), epigallcatechin 3-gallate (EGCg), epiafzelechin, fisetinidol,gallocatechin (GC), gallcatechin 3-gallate (GCg), guibourtinidol,mesquitol, robinetinidol, theaflavin-3-gallate, theaflavin-3′-gallate,theflavin-3,3′-digallate, thearubigin), flavan-4-ols (e.g., apiforoland/or luteoforol) and/or flavan-3,4-diols (e.g., leucocyanidin,leucodelphinidin, leucofisetinidin, leucomalvidin, luecopelargonidin,leucopeonidin, leucorobinetinidin, melacacidin and/or teracacidin);and/or

one or more isoflavonoids, optionally one or more isoflavones, such asbiochanin A, daidzein, formononetin, genistein and/or glycitein;isoflavanes, such as equol, ionchocarpane and/or laxifloorane;isoflavandiols; isoflavenes, such asglabrene, haginin D and/or2-methoxyjudaicin; coumestans, such as coumestrol, plicadin and/orwedelolactone; pterocarpans; and/or roetonoids; and/or

one or more neoflavonoids, optionally calophyllolide, coutareagenin,dalbergichromene, dalbergin and/or nivetin; and/or

one or more pterocarpans, optionally bitucarpin A, bitucarpin B,erybraedin A, erybraedin B, erythrabyssin II, erthyrabissin-1,erycristagallin, glycinol, glyceollidins, glyceollins, glycyrrhizol,maackiain, medicarpin, morisianine, orientanol, phaseolin, pisatin,striatine and/or trifolirhizin.

Paragraph [13]. The method of any one of paragraphs 1-12, wherein thecomposition further comprises one or more water-soluble anionicsurfactants and/or one or more water-insoluble anionic surfactants,optionally: one or more anionic surfactants selected from the groupconsisting of alkyl carboxylates (e.g., sodium stearate), alkyl sulfates(e.g., alkyl lauryl sulfate, sodium lauryl sulfate), alkyl ethersulfates, alkyl amido ether sulfates, alkyl aryl polyether sulfates,alkyl aryl sulfates, alkyl aryl sulfonates, alkyl sulfonates, alkylamide sulfonates, alkyl aryl sulfonates, alkyl benzene sulfonates, alkyldiphenyloxide sulfonate, alpha-olefin sulfonates, alkyl naphthalenesulfonates, paraffin sulfonates, alkyl sulfosuccinates, alkyl ethersulfosuccinates, alkylamide sulfosuccinates, alkyl sulfosuccinamates,alkyl sulfoacetates, alkyl phosphates, alkyl ether phosphates, acylsarconsinates, acyl isethionates, N-acyl taurates,N-acyl-N-alkyltaurates, benzene sulfonates, cumene sulfonates, dioctylsodium sulfosuccinate, ethoxylated sulfosuccinates, lignin sulfonates,linear alkylbenzene sulfonates, monoglyceride sulfates,perfluorobutanesulfonate, perfluorooctanesulfonate, phosphate ester,styrene acrylic polymers, toluene sulfonates and xylene sulfonates.

Paragraph [14]. The method of any of one of paragraphs 1-13, wherein thecomposition further comprises one or more cationic surfactants,optionally: alkyltrimethylammonium salts (e.g., cetyl trimethylammoniumbromide, cetyl trimethylammonium chloride), cetylpyridinium chloride,benzalkonium chloride, benzethonium chloride,5-Bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride,cetrimonium bromide, dioctadecyldimethylammonium bromide and/oroctenidine dihydrochloride.

Paragraph [15]. The method of any one of paragraphs 1-14, wherein thecomposition further comprises one or more water-soluble nonionicsurfactants and/or one or more water-insoluble nonionic surfactants,optionally: alcohol ethoxylates, alkanolamides, alkanolaminecondensates, carboxylic acid esters, cetostearyl alcohol, cetyl alcohol,cocamide DEA, dodecyldimethylamine oxides, ethanolamides, ethoxylates ofglycerol ester and glycol esters, ethylene oxide polymers, ethyleneoxide-propylene oxide copolymers, glucoside alkyl ethers, glycerol alkylethers, glycerol esters, glycol alkyl ethers (e.g., polyoxyethyleneglycol alkyl ethers, polyoxypropylene glycol alkyl ethers), glycolalkylphenol ethers (e.g., polyoxyethylene glycol alkylphenol ethers,),glycol esters, monolaurin, pentaethylene glycol monododecyl ethers,poloxamer, polyamines, polyglycerol polyricinoleate, polysorbate,polyoxyethylenated fatty acids, polyoxyethylenated mercaptans,polyoxyethylenated polyoxyproylene glycols, polyoxyethylene glycolsorbitan alkyl esters, polyethylene glycol-polypropylene glycolcopolymers, polyoxyethylene glycol octylphenol ethers, polyvinylpynolidones, sugar-based alkyl polyglycosides, sulfoanylamides, sorbitanfatty acid alcohol ethoxylates, sorbitan fatty acid ester ethoxylates,sorbitan fatty acid ester and/or tertiary acetylenic glycols.

Paragraph [16]. The method of any one of paragraphs 1-15, wherein thecomposition further comprises one or more zwitterionic surfactants,optionally: 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate,cocamidopropyl betaine, cocamidopropyl hydroxysultaine,phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and/orone or more sphingomyelins.

Paragraph [17]. The method of any one of paragraphs 1-16, wherein thecomposition further comprises one or more soaps and/or organosiliconesurfactants, optionally: one or more alkali metal salts of fatty acids.

Paragraph [18]. A method of any one of paragraphs 1-9, wherein thecomposition comprises a granule comprising one or more enzymes,optionally wherein the granule has an average particle size of 20-2000um equivalent spherical diameter.

Paragraph [19]. A method of paragraph 18, wherein the compositioncomprises a core and optionally one or more coatings surrounding thecore, wherein the core optionally comprises:

one ore more additional materials such as fillers, fibre materials(cellulose or synthetic fibres), stabilizing agents, solubilizingagents, suspension agents, viscosity regulating agents, light spheres,plasticizers, salts, lubricants and fragrances;

one or more binders, such as synthetic polymer, wax, fat, orcarbohydrate;

one ore more of a salt of a multivalent cation, a reducing agent, anantioxidant, a peroxide decomposing catalyst and/or an acidic buffercomponent, optionally as a homogenous blend;

one ore more of an inert particle with an enzyme absorbed into it, orapplied onto the surface, for example, by fluid bed coating.

Paragraph [20]. A method comprising foliar application of a compositioncomprising a phytoprotective agent to foliage of a plant, wherein saidphytoprotective agent comprises a protease, wherein said compositionfurther comprises an agriculturally acceptable carrier, wherein saidcomposition is applied an effective amount to control one or morediseases or pests of the plant, induce disease resistance or pestresistance of the plant, improve plant health, reduce disease severity,reduce pathogenic infection, or a combination thereof.

Paragraph [21]. The method of any one of paragraphs 1-20, wherein thedisease severity is reduced by at least about 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% comparedto a plant that has not received application of the composition.

EXAMPLES

The following examples are not intended to be a detailed catalogue ofall the different ways in which the present disclosure may beimplemented or of all the features that may be added to the presentdisclosure. Subjects skilled in the art will appreciate that numerousvariations and additions to the various embodiments may be made withoutdeparting from the present disclosure. Hence, the following descriptionsare intended to illustrate some particular embodiments of the inventionand not to exhaustively specify all permutations, combinations andvariations thereof.

In the following examples, Composition 2 comprises a protease having thepolypeptide sequence of SEQ ID NO: 1. Composition 4 comprises a proteasehaving the polypeptide sequence of SEQ ID NO: 2. Composition 5 comprisesa protease having the polypeptide sequence of SEQ ID NO: 3. Composition6 comprises an amylase, used as a negative control. Composition 7comprises a protease having the polypeptide sequence of SEQ ID NO: 4.Composition 8 comprises a protease having the polypeptide sequence ofSEQ ID NO: 5.

Example 1

Inoculum Preparation: Infected tomato leaves from inoculated plants wereplaced in a plastic bag with a wet paper towel and incubated on agreenhouse bench at 18° C. for 24 hours. The leaves were placed in asterile flask and rinsed with cold sterile distilled water. The sporesuspension was filtered through 2 layers of cheesecloth into anothersterile flask. The sporangia concentration of the inoculum wascalculated using a haemocytometer and diluted with cold steriledistilled water to 10,000 sporangia per mL. The inoculum was placed intoa fridge (4° C.) for 0.5-1 hour. The inoculum was removed from thefridge and allowed to warm up to room temperature for 0.5-1 hour toinduce zoospore release.

Treatment Application and Inoculation of Tomato Plants: Six week oldcherry tomato plants were used. There were 4 plants per treatment.Treatment plants were sprayed until run off with a 0.5% solution ofComposition 2, or water, in a container and placed on a greenhouse benchfor 24 hours, after which certain plants were sprayed until run off witha spore suspension of Phytophthora infestans (10,000 sporangia/ml) orwater. They were then placed in a humidity chamber in the dark at 18° C.with 100% relative humidity for 48 hours. The plants were removed fromthe humidity chamber, placed on a greenhouse bench and maintained to 18°C. After 5-7 days the plants were visually rated for disease severity.

TABLE 1 Percent disease severity and disease reduction Treatment DiseaseSeverity (%)^(X) Disease Reduction (%) 1 P. infestans 67.5^(A)  — 2 P.infestans +  8.75^(B) 87 Composition 2 3 Composition 2 0^(B )  — 4 Water0^(B )  — ^(X)-Means separation based on Tukey-Kramer HSD. Means withthe same letter are not significantly different.

Example 2

Inoculum Preparation: Infected tomato leaves from inoculated plants wereplaced in a plastic bag with a wet paper towel and incubated on agreenhouse bench at 18° C. for 24 hours. The leaves were placed in asterile flask and rinsed with cold sterile distilled water. The sporesuspension was filtered through 2 layers of cheesecloth into anothersterile flask. The sporangia concentration of the inoculum wascalculated using a haemocytometer and diluted with cold steriledistilled water to 10,000 sporangia per mL. The inoculum was placed intoa fridge (4° C.) for 0.5-1 hour. The inoculum was removed from thefridge and allowed to warm up to room temperature for 0.5-1 hour toinduce zoospore release.

Treatment Application and Inoculation of Tomato Plants: Six week oldcherry tomato plants were used. There were 4 plants per treatment.Treatment plants were sprayed until run off with a 0.5% solution ofComposition 2, or water, in a container and placed on a greenhouse benchfor 24 hours, after which certain plants were sprayed until run off witha spore suspension of P. infestans (10,000 sporangia/ml) or water. Theywere then placed in a humidity chamber in the dark at 18° C. with 100%relative humidity for 48 hours. The plants were removed from thehumidity chamber, placed on a greenhouse bench and maintained to 18° C.After 5-7 days the plants were visually rated for disease severity.

TABLE 2 Percent disease severity and disease reduction Treatment DiseaseSeverity (%)^(X) Disease Reduction (%) 1 P. infestans 45^(A)   — 2 P.infestans + 21.3^(B) 52.8 Composition 2 3 Composition 2 0^(B)  — 4 Water0^(B)  — ^(X)-Means separation based on Tukey-Kramer HSD. Means with thesame letter are not significantly different.

Example 3

Inoculum Preparation: Infected tomato leaves from inoculated plants wereplaced in a plastic bag with a wet paper towel and incubated on agreenhouse bench at 18° C. for 24 hours. The leaves were placed in asterile flask and rinsed with cold sterile distilled water. The sporesuspension was filtered through 2 layers of cheesecloth into anothersterile flask. The sporangia concentration of the inoculum wascalculated using a haemocytometer and diluted with cold steriledistilled water to 10,000 sporangia per mL. The inoculum was placed intoa fridge (4° C.) for 0.5-1 hour. The inoculum was removed from thefridge and allowed to warm up to room temperature for 0.5-1 hour toinduce zoospore release.

Treatment Application and Inoculation of Tomato Plants: Six week oldcherry tomato plants were used. There were 4 plants per treatment.Treatment plants were sprayed until run off with a 0.3% solution ofComposition 2, a 0.3% solution of Composition 2 combined with asurfactant, nothing, or water, in a container and placed on a greenhousebench for 24 hours, after which certain plants were sprayed until runoff with a spore suspension of P. infestans (10,000 sporangia/ml) orwater. They were then placed in a humidity chamber in the dark at 18° C.with 100% relative humidity for 48 hours. The plants were removed fromthe humidity chamber, placed on a greenhouse bench and maintained to 18°C. After 5-7 days the plants were visually rated for disease severity.

TABLE 3 Percent disease severity and disease reduction Treatment DiseaseSeverity (%)^(X) Disease Reduction (%) 1 P. infestans 90^(A)   — 2 P.infestans + 13.8^(B) 84.7 Composition 2 3 P. infestans +   3.1^(CD) 96.5Composition 2 + surfactant 4 Composition 2 0^(D)   — 5 Water 0^(D)   —^(X)-Means separation based on Tukey-Kramer HSD. Means with the sameletter are not significantly different.

Example 4

Inoculum Preparation: 3 days prior to inoculation, glycerol stocks ofPseudomonas syringae were streaked onto Pseudomonas selective media andincubated at 30° C. for 48 hours. 24 hours prior to inoculation, singlecolonies of P. syringae were collected from the selective media andplaced into 100 mL of LB media. The inoculated LB media was placed intoa shaker incubator at 28° C. and 200 RPM overnight. The flasks wereremoved just prior to application, and 5 mL of Pst was transferred to 45mL of 10 mM MgCl2 and shaken. Using a spectrophotometer, the absorptionat OD₆₀₀ was taken. This was used to calculate the appropriate level ofdilution to achieve OD₆₀₀=0.03. 200 mL of Pst solution at OD₆₀₀=0.03were prepared, and 40 μL of Silwet was added to the inoculum.

Treatment Application and Inoculation of Tomato Plants: Seven tomatoplants at four weeks old were selected per treatment. Treatment plantswere sprayed until run-off with 0.5% Composition 2 or water. The plantswere placed into a humidity chamber (RH 100%) for 24 hours attemperatures of 24° C. daytime and 20° C. night. 24 hours later certainplants were sprayed until runoff with a bacterial suspension of P.syringae (OD₆₀₀=0.03) or 10 mM MgCl₂. They were then placed in ahumidity chamber (RH 100%) at 24° C. daytime and 20° C. nighttemperatures for 24 hours. The plants were removed from the humiditychamber, placed on a greenhouse bench and maintained at the sametemperatures. After 5 days the 10 most severely infected leaves weresampled, scanned and rated for disease severity using ASSESS imageanalysis software.

TABLE 4 Percent disease severity and disease reduction Treatment DiseaseSeverity (%)^(X) Disease Reduction (%) 1 P. syringae ^(Y) 100.00^(A) — 2P. syringae +  11.08^(B) 88.93 Composition 2 3 Composition 2 0^(C)  — 4MgCl₂ 0^(C)  — ^(X)-Means separation based on Tukey-Kramer HSD. Meanswith the same letter are not significantly different. ^(Y)-Markedtreatment had >10 leaves that could not be scanned due to leaf death andwere rated 100.00% severity.

Example 5

Inoculum Preparation: 3 days prior to inoculation, glycerol stocks of P.syringae were streaked onto Pseudomonas selective media and incubated at30° C. for 48 hours. 24 hours prior to inoculation, single colonies ofP. syringae were collected from the selective media and placed into 100mL of LB media. The inoculated LB media was placed into a shakerincubator at 28° C. and 200 RPM overnight. The flasks were removed justprior to application, and 5 mL of Pst was transferred to 45 mL of 10 mMMgCl₂ and shaken. Using a spectrophotometer, the absorption at OD₆₀₀ wastaken. This was used to calculate the appropriate level of dilution toachieve OD₆₀₀=0.03 200 mL of Pst solution at OD₆₀₀=0.03 were prepared,and 40 μL of Silwet was added to the inoculum.

Treatment Application and Inoculation of Tomato Plants: Seven tomatoplants at four weeks old were selected per treatment. Treatment plantswere sprayed until run-off with 0.5% Composition 2 or water. The plantswere placed into a humidity chamber (RH 100%) for 24 hours attemperatures of 24° C. daytime and 20° C. night. 24 hours later certainplants were sprayed until runoff with a bacterial suspension of P.syringae (OD₆₀₀=0.03) or 10 mM MgCl₂. They were then placed in ahumidity chamber (RH 100%) at 24° C. daytime and 20° C. nighttemperatures for 24 hours. The plants were removed from the humiditychamber, placed on a greenhouse bench and maintained at the sametemperatures. After 5 days the 10 most severely infected leaves weresampled, scanned and rated for disease severity using Assess imageanalysis software.

TABLE 5 Percent disease severity and disease reduction Treatment DiseaseSeverity (%)^(X) Disease Reduction (%) 1 P. syringae ^(Y) 100.00^(A) — 2P. syringae +  9.2^(C) 90.8 Composition 2 3 Composition 2 0^(C)  — 4MgCl₂ 0^(C)  — ^(X)-Means separation based on Tukey-Kramer HSD. Meanswith the same letter are not significantly different. ^(Y)-Markedtreatment had >10 leaves that could not be scanned due to leaf death andwere rated 100.00% severity.

Example 6

Inoculum Preparation: 3 days prior to inoculation, glycerol stocks of P.syringae were streaked onto Pseudomonas selective media and incubated at30° C. for 48 hours. 24 hours prior to inoculation, single colonies ofP. syringae were collected from the selective media and placed into 100mL of LB media. The inoculated LB media was placed into a shakerincubator at 28° C. and 200 RPM overnight. The flasks were removed justprior to application, and 5 mL of Pst was transferred to 45 mL of 10 mMMgCl2 and shaken. Using a spectrophotometer, the absorption at OD₆₀₀ wastaken. This was used to calculate the appropriate level of dilution toachieve OD₆₀₀=0.03 200 mL of Pst solution at OD₆₀₀=0.03 were prepared,and 40 μL of Silwet was added to the inoculum.

Treatment Application and Inoculation of Tomato Plants: Seven tomatoplants at four weeks old were selected per treatment. Treatment plantswere sprayed until run-off with 0.3% Composition 2, 0.3% Composition 2combined with a surfactant, or water. The plants were placed into ahumidity chamber (RH 100%) for 24 hours at temperatures of 24° C.daytime and night. 24 hours later certain plants were sprayed untilrunoff with a bacterial suspension of P. syringae (OD₆₀₀=0.03) or 10 mMMgCl2. They were then placed in a humidity chamber (RH 100%) at 24° C.daytime and 20° C. night temperatures for 24 hours. The plants wereremoved from the humidity chamber, placed on a greenhouse bench andmaintained at the same temperatures. After 5 days the 10 most severelyinfected leaves were sampled, scanned and rated for disease severityusing ASSESS™ image analysis software.

TABLE 6 Percent disease severity and disease reduction Treatment DiseaseSeverity (%)^(X) Disease Reduction (%) 1 P. syringae 15.0^(A) — 2 P.syringae +  9.2^(B) 39.2 Composition 2 3 P. syringae +  2.9^(C) 80.9Composition 2 + surfactant 4 Composition 2 0^(C)  — 5 Water 0^(C)  —^(X)-Means separation based on Tukey-Kramer HSD. Means with the sameletter are not significantly different.

Example 7

Inoculum Preparation: 7 days prior to the treatment, 2 cherry tomatoplants were inoculated with P. infestans from 10-day-old Rye A+ plates.24 hours prior to treatment, infected tomato leaves from the inoculatedplants were placed in a plastic bag with a moist paper towel. Theinfected leaves were left on greenhouse bench at 18° C. for 24 hours.The leaves were placed in a sterile flask and rinsed with cold steriledistilled water. The spore suspension was then filtered through 2 layersof cheesecloth into another sterile flask. The sporangia concentrationwas calculated using haemocytometer. The inoculum concentration wasadjusted to 10,000 sporangia per mL using cold sterile distilled waterand inoculum was placed in fridge (4° C.) for 0.5-1 hour. After 1 hourthe spore suspension was warmed to room temperature (0.5-1 hour) toinduce zoospore release.

Treatment Application and Assay: Leaf disks were cut from leavesselected from 9-10 week old cherry tomato plants. Disks were dipped in0.3% Composition solutions or water for 30 seconds. The disks wereplaced adaxial side up in each of the wells on plates. Each of the 24wells contained 2 ml of 0.5% water agar. 2 plates of each treatment wereprepared for a total of 48 leaf disks per treatment. 1-3 hours aftertreatment, the disks were wounded in the center with a sterile needleand 10 μL of P. infestans spore suspension or sterile water was droppedinto the center of the disk. The plates were sealed with Parafilm andwrapped in foil. The wrapped plates were placed in an 18° C. incubatorfor 2 days. After 2 days, the foil was removed from the plates and theywere incubated at 18° C. under light (16 hours' cool white fluorescentlight and 8 hours dark). Disease severity was assessed after 5 daysusing the visible spectrum settings of a LEMNATEC™ SCANALYZER™.

TABLE 7 Percent healthy tissue, diseased tissue, and disease reduction,visible spectrum analysis Disease Treatment Healthy (%)^(X) Disease(%)^(X) Reduction (%) 1 P. infestans  34.3^(D) 65.7^(A) — 2 P.infestans +  62.1^(C) 37.9^(B) 42.2 Composition 2 3 P. infestans + 81.8^(AB)  18.2^(CD) 72.3 Composition 4 4 P. infestans +  71.9^(BC) 28.1^(BC) 57.2 Composition 5 5 P. infestans +  28.2^(D) 71.8^(A) −9.3Composition 6 6 Water  91.3^(A)  8.7^(D) — ^(X)-Means separation basedon Tukey-Kramer HSD. Means with the same letter are not significantlydifferent.

Example 8

Inoculum Preparation: 7 days prior to the treatment, 2 cherry tomatoplants were inoculated with P. infestans from 10-day-old Rye A+ plates.24 hours prior to treatment, infected tomato leaves from the inoculatedplants were placed in a plastic bag with a moist paper towel. Theinfected leaves were left on greenhouse bench at 18° C. for 24 hours.The leaves were placed in a sterile flask and rinsed with cold steriledistilled water. The spore suspension was then filtered through 2 layersof cheesecloth into another sterile flask. The sporangia concentrationwas calculated using haemocytometer. The inoculum concentration wasadjusted to 10,000 sporangia per mL using cold sterile distilled waterand inoculum was placed in fridge (4° C.) for 0.5-1 hour. After 1 hourthe spore suspension was warmed to room temperature (0.5-1 hour) toinduce zoospore release.

Treatment Application and Assay: Leaf disks were cut from leavesselected from 9-10 week old cherry tomato plants. Disks were dipped in0.3% Composition solutions or water for 30 seconds. The disks wereplaced adaxial side up in each of the wells on plates. Each of the 24wells contained 2 ml of 0.5% water agar. 2 plates of each treatment wereprepared for a total of 48 leaf disks per treatment. 1-3 hours aftertreatment, the disks were wounded in the center with a sterile needleand 10 μL of P. infestans spore suspension or sterile water was droppedinto the center of the disk. The plates were sealed with Parafilm andwrapped in foil. The wrapped plates were placed in an 18° C. incubatorfor 2 days. After 2 days, the foil was removed from the plates and theywere incubated at 18° C. under light (16 hours' cool white fluorescentlight and 8 hours dark). Disease severity was assessed after 5 daysusing the visible spectrum settings of a LEMNATEC™ SCANALYZER™.

TABLE 8 Percent healthy tissue, diseased tissue, and disease reduction,visible spectrum analysis Disease Treatment Healthy (%) Disease (%)Reduction (%) 1 P. infestans 29.5^(E) 70.5^(A) — 2 P. infestans +77.6^(B) 22.4^(D) 68.3 Composition 2 3 P. infestans +  82.2^(AB) 17.8^(DE) 74.7 Composition 4 4 P. infestans +  76.3^(BC)  23.7^(CD)66.4 Composition 5 5 P. infestans + 33.4^(E) 66.6^(A)  5.5 Composition 66 P. infestans +   64.6^(CD)  35.4^(BC) 49.7 Composition 7 7 P.infestans + 57.1^(D) 42.9^(B) 39.1 Composition 8 8 Water 90.5^(A) 9.5^(E) — ^(X)-Means separation based on Tukey-Kramer HSD. Means withthe same letter are not significantly different.

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodtherefrom as modifications will be obvious to those skilled in the art.It is not an admission that any of the information provided herein isprior art or relevant to the presently claimed inventions, or that anypublication specifically or implicitly referenced is prior art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

That which is claimed:
 1. A composition comprising a polypeptide in anagriculturally acceptable carrier, said polypeptide having proteolyticactivity and comprising an amino acid sequence that is at least 95%identical to one or more of SEQ ID NOs: 1-5.
 2. The composition of claim1, further comprising one or more pesticides.
 3. The composition ofclaim 1, further comprising one or more surfactants.
 4. The compositionof claim 1, further comprising one or more nonionic surfactants. Thecomposition of claim 1, wherein said polypeptide comprises an amino acidsequence that is at least 96% identical to one or more of SEQ ID Nos:1-5.
 6. The composition of claim 1, wherein said polypeptide comprisesan amino acid sequence that is at least 97% identical to one or more ofSEQ ID Nos: 1-5.
 7. The composition of claim 1, wherein said polypeptidecomprises an amino acid sequence that is at least 98% identical to oneor more of SEQ ID Nos: 1-5.
 8. The composition of claim 1, wherein saidpolypeptide comprises an amino acid sequence that is at least 99%identical to one or more of SEQ ID Nos: 1-5.
 9. The composition of claim1, wherein said polypeptide comprises SEQ ID NO:
 1. 10. The compositionof claim 1, wherein said polypeptide comprises SEQ ID NO:
 2. 11. Thecomposition of claim 1, wherein said polypeptide comprises SEQ ID NO: 3.12. The composition of claim 1, wherein said polypeptide comprises SEQID NO:
 4. 13. The composition of claim 1, wherein said polypeptidecomprises SEQ ID NO:
 5. 14. The composition of claim 1, wherein saidpolypeptide comprises a functional proteolytic fragment of SEQ ID NO: 1.15. The composition of claim 1, wherein said polypeptide comprises afunctional proteolytic fragment of SEQ ID NO:
 2. 16. The composition ofclaim 1, wherein said polypeptide comprises a functional proteolyticfragment of SEQ ID NO:
 3. 17. The composition of claim 1, wherein saidpolypeptide comprises a functional proteolytic fragment of SEQ ID NO: 4.18. The composition of claim 1, wherein said polypeptide comprises afunctional proteolytic fragment of SEQ ID NO:
 5. 19. A method comprisingapplying the composition of claim 1 to a plant and/or plant part.
 20. Amethod comprising applying the composition of claim 1 to a plant growthmedium.